Live, attenuated strains of SIV still afford the most reliable protection against pathogenic challenge viruses in animal models. Thus, a better understanding of the mechanisms of protection by attenuated viruses may provide insights crucial to the development of a safe and effective AIDS vaccine. We have developed an approach for producing genetically engineered strains of SIV that are limited to a single round of infection as a non-replicating AIDS vaccine approach. Single-cycle SIV (scSIV)-infected cells express all of the viral gene products except for Pol and release immature virus particles that cannot complete subsequent rounds of infection. In previous studies, rhesus macaques immunized with scSIV made diverse virus-specific immune responses and were able to partially contain viral loads after an intravenous challenge with wild-type SIVmac239. More recently, we evaluated the effects of trans-complementation with the vesicular stomatitis virus glycoprotein (VSV G) and the route of administration on virus-specific T cell responses. After a single dose of VSV G trans-complemented scSIV (VSV G scSIV), SIV-specific CD8+ T cell responses were more than 10-fold higher than previous responses in animals inoculated with non-trans-complemented scSIV. Phenotypic analysis of virus-specific CD8+ T cells also revealed differences in the expression of the mucosal homing receptor a4p7 depending on the route of inoculation. Here we propose to use VSV G scSIV as a tool to further investigate mechanisms of mucosal immunity and the extent of heterologous protection that may be achieved by this vaccine approach. For specific aim 1, we will test the hypothesis that the site of immunization with VSV G scSIV determines the mucosal homing properties of virus-specific T cells and resistance to an intrarectal challenge with SIVmac239. For specific aim 2, we will test the hypothesis that the site of priming influences the homing of virus-specific T cell responses expanded after a systemic boost with VSV G scSIV and the degree of protection against a vaginal challenge with SIVmac239. For specific aim 3, we will test the hypothesis that immunization with a mixture of antigenically divergent strains of VSV G scSIV can broaden virus-specific immune responses and enhance protection against a heterologous challenge virus. These studies contribute to the overall goals of this program project to define mechanisms of protective immunity by attenuated vaccine strains and to pursue promising new AIDS vaccine concepts.